Human herpesvirus 7 meningitis in an adolescent with normal immune function:A case report

BACKGROUND Human herpesvirus type 7(HHV-7)is a less common herpes virus that usually causes mild,self-limiting illnesses.However,in recent years,there have been increasing reports of HHV-7 causing serious central nervous system infections,especially meningitis.The pathogenesis and clinical features of HHV-7 meningitis,particularly in adolescents with normal immune function,remain incompletely studied.Therefore,the purpose of this report is to share a case of HHV-7 meningitis in an immunocompetent adolescent with a view to deepening our understanding of the disease.CASE SUMMARY A 12-year-old female was admitted with fever,headache,and vomiting.4 d before admission,the patient developed a fever without obvious induction,with a temperature up to 39.5℃,no convulsions,accompanied by chills,headaches,fatigue,and no muscle aches.The patient was treated with fever reduction,which could be reduced to 38℃;repeated high fever,accompanied by vomiting 7-8 times;and no abdominal pain or diarrhea.The patient was diagnosed with"acute suppurative tonsillitis"in a local hospital,and the blood routine was generally normal.The patient was given symptomatic support treatment such as"ceftriaxone sodium"and antiemetic rehydration for 2 d,and his condition did not improve.The patient's physical examination showed pharyngeal congestion,bilateral tonsil grade I hypertrophy,regression of purulent secretions,and cervical resistance.Ocular B-ultrasound:Opacity of the vitreous body and edema of the optic disc in both eyes.Optical coherence tomography examination showed that the macular fovea was generally normal in both eyes,with edema of the optic disc.DNA virus monitoring results:HHV-7.We gave ganciclovir antiviral therapy,dexamethasone anti-inflammatory treatment,mannitol to reduce cranial pressure,omeprazole to protect gastrointestinal mucosa,and calcium and potassium supplementation.CONCLUSION This study reports a case of HHV-7 meningitis in an adolescent with normal immune function.Through comprehensive analysis of the clinical manifestations,laboratory tests,and treatment methods of the patient,it is found that early identification and antiviral treatment are essential for the outcome of the disease.This case suggests that despite normal immune function,adolescents may still suffer from herpes virus type 7 meningitis,so clinicians should be vigilant and take effective treatment measures in time.

Genotypic analysis on the ORF-K1 gene of human herpesvirus 8 from patients with Kaposi's sarcoma in Xinjiang,China

Human herpesvirus 8 （HHV-8） is thought to be essential for the development of all forms of Kaposi＇s sarcoma （KS）. HHV-8 DNA is present virtually in all KS tumor biopsy samples. Genes at both ends of the HHV-8 genome have been shown to vary considerably. Seven major molecular subtypes of HHV-8 were defined based on the amino acid sequence of the open reading frame K1 （ORF-K1）, generally known as A, B, C, D, E, F, and Z. Most strains collected worldwide were clustered into two subtypes （A and C）. Here, the K1/VRI region of HHV-8 was amplified by nested PCR in 22 （81.48%） of 27 cases from Xinjiang Uygur Autonomous Region, a province in northwestern China. Phylogenetic analysis on the basis of the K1/VR1 amino acid sequence indicated that the majority of these KS patients were infected by subtype C HHV-8 （n = 18, including 15 belonging to the C2 group）, and several by subtype A （n = 4, including 3 being the A1 group）. This is the first report of subtype A HHV-8 in China. Furthermore, the correlations between different forms and lesions of KS and different subtypes of HHV-8 were analyzed. The findings showed that subtype A HHV-8 resulted in significantly more frequent mucosal KS lesions than subtype C. However, there was no obvious correlation between different forms of KS and different subtypes of HHV-8.

Establishment of an ELISA to Detect Kaposi's Sarcoma-associated Herpesvirus Using Recombinant ORF73

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and a proportion of cases of multicentric Castleman's disease (MCD). The ORF73 protein was cloned into pQE80L-orf73 and expressed in E.coli and purified. The expressed recombinant ORF73 was identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). A protein of about 27 kDa was expressed as expected. Western Blotting showed that the purified recombinant ORF73 reacted with KSHV positive serum. The immunogenicity of the recombinant ORF73 was further analysed by ELISA and the optimal conditions were determined. The ORF73 ELISA was used to compare the KSHV seroprevalence between Hubei and Xinjiang Han people. The Han people in Xinjiang have significantly higher KSHV seroprevalence than their counterparts in Hubei (6.7% vs 2.9%, P = 0.005).

Isolation and Identification of a Specific cDNA Mapping to the Bam HI-I2 and -LFragments within the Inverted Repeats ofUnique Long Re-gion (IRL) in the Genom e ofMarek′s Disease Herpesvirus (MDV) Oncogenic Strain Beijing-1

Objective\ To understand the transcription of BamHI L DNA fragment from genome of strong virulent GA strain of Marek′s disease herpesvirus (MDV) in lymphoblastoid tumor tissue induced by oncogenic strain Beijing 1 (a specific local strain in China) of MDV. Methods\ Two oligonucleotide primers were synthesized according to the reported sequence of \%meq\% gene an ideal oncogenic candidate and our previously determined sequence of BamHI L fragment of Marek′s disease herpesvirus (MDV), respectively. Reverse transcriptase PCR(RT PCR) assay was performed by using these primers and the mRNA as a template which was isolated from visceral lymphoblastoid tumors obtained from chickens artificially infected with strain Beijing 1 of oncogenic MDV. Southern blot molecular hybridization was further carried out to detect the product of RT PCR with digoxigenin labeled nucleotide probe from BamHI I2 and L fragment in the gene library of MDV strain GA, respectively. Results\ Two probes could simultaneously hybridize this cDNA amplified by RT PCR with a length of about 730 bp. Conclusion\ It is suggested that \%meq\% transcription could extend from the right hand end of BamHI I2 to the adjacent BamHI L, and the BamHI L region was likely to be transcribed in MDV induced lymphoblastoid tumors.

Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi's Sarcoma Associated Herpesvirus orf65

To purify the protein encoding the small capsid protein （SCP） of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi＇s sarcoma associated-herpesvirus （KSHV） was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-13-D-thiogalactopyranoside （IPTG） and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis （SDS-PAGE） and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.

Expression of Kaposi's Sarcoma-associated Herpesvirus ORFK8.1 and Its Preliminary Diagnostic Application

The ORFK8.1 of Kaposi＇s sarcoma associated-herpesvirus （KSHV） was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf KS.1 was induced by isopropyl-b-D-thiogalactopyranoside （IPTG）. The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis （SDS-PAGE）. SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant ORFKS. 1 protein. The optimal condition of the recombinant ORFKS. 1 ELISA assay was confirmed： the concentration of antigen was 5 μg/mL, the dilution of serum was 1：200. We used the ELISA method to investigate the recombinant ORF KS. 1 protein＇s specificity, the data showed that the specificity of ORF KS.1 to detect KSHV was 100%. At the same time, 560 sera samples from Hubei province were detected by using ORFKS. 1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.

Molecular and in vitro Characterization of Field Isolates of Bovine Herpesvirus-1

Bovine Herpesvirus-1 (BoHV-1) is distributed worldwide and is a major pathogen in cattle, being the causal agent of a variety of clinical syndromes. The aim of this study was to isolate and to characterize (molecular and biological characterization) BoHV-1 from 29 immunosuppressed animals. It was possible to obtain 18 isolates, each from a different animal, such as from the respiratory and reproductive tracts. In some cases the cytopathic effect was visible 12 hours post-inoculation, and became characteristic after 36-48 hours. Biological characteristics were evaluated and compared with Iowa and Colorado-1 reference strains, and differences were found in plaque size, virus titer measured by TCID50 and PFU/mL, and one step virus curves. These results showed that some isolates had a highly virulent-like behavior in vitro, compared to the reference strains, with shorter eclipse periods, faster release of virus into the supernatants, and higher burst size and viral titer. There were no differences in glycoprotein expression of BoHV-1 isolates, measured by Western blot on monolayers. Moreover, using restriction endonucleases analysis, most of the viruses were confirmed as BoHV-1.1 and just one of them was confirmed as BoHV-1.2a subtype. These findings suggest that some wild-type BoHV-1 isolates could be useful as seeds to develop new monovalent vaccines.

Contributions of neurotropic human herpesviruses herpes simplex virus 1 and human herpesvirus 6 to neurodegenerative disease pathology

Human herpesviruses （HVs） have developed ingenious mechanisms that enable them to traverse the defenses of the central nervous system （CNS）. The ability of HVs to enter a state of latency, a defining char- acteristic of this viral family, allows them to persist in the human host indefinitely. As such, HVs represent the most frequently detected pathogens in the brain. Under constant immune pressure, these infections are largely asymptomatic in healthy hosts. However, many neurotropic HVs have been directly connected with CNS pathology in the context of other stressors and genetic risk factors. In this review, we discuss the potential mechanisms by which neurotropic HVs contribute to neurodegenerative disease （NDD） patholo- gy by highlighting two prominent members of the HV family, herpes simplex virus 1 （HSV-1） and human herpesvirus 6 （HHV-6）. We （i） introduce the infectious pathways and replicative cycles of HSV-1 and HHV-6 and then （ii） review the clinical evidence supporting associations between these viruses and the NDDs Alzheimer＇s disease （AD） and multiple sclerosis （MS）, respectively. We then （iii） highlight and dis- cuss potential mechanisms by which these viruses exert negative effects on neurons and glia. Finally, we （iv） discuss how these viruses could interact with other disease-modifying factors to contribute to the initiation and/or progression of NDDs.

The Importance of Heparan Sulfate in Herpesvirus Infection

Herpes simplex virus type-1 (HSV-1) is one of many pathogens that use the cell surface glycosaminoglycan heparan sulfate as a receptor. Heparan sulfate is highly expressed on the surface and extracellular matrix of virtually all cell types making it an ideal receptor. Heparan sulfate interacts with HSV-1 envelope glycoproteins gB and gC during the initial attachment step during HSV-1 entry. In addition,a modified form of heparan sulfate,known as 3-O-sulfated heparan sulfate,interacts with HSV-1 gD to induce fusion between the viral envelope and host cell membrane. The 3-O-sulfation of heparan sulfate is a rare modification which occurs during the biosynthesis of heparan sulfate that is carried out by a family of enzymes known as 3-O-sulfotransferases. Due to its involvement in multiple steps of the infection process,heparan sulfate has been a prime target for the development of agents to inhibit HSV entry. Understanding how heparan sulfate functions during HSV-1 infection may not only be critical for inhibiting infection by this virus,but it may also be crucial in the fight against many other pathogens as well.

Reactivation of Latent Infection of Human Herpesvirus 8 in BC-3 Cells from Primary Effusion Lymphoma by Recombinant CytokinesSimilar to that Produced by HIV-1-infected T Cells

Objective: To study and confirm that recombinant cytokines similar to those produced by HIV-1 infected T cells induced lytic cycle replication of human herpesvirus 8 (HHV-8) in BC-3 cells, another cell line from primary effusion lymphama(PEL). Methods: The persistent stimulation of BC-3 was conducted by several cytokines known to be produced by HIV-1-infected T cells and important in growth and proliferation of Kaposi's sarcoma(KS)cells in vitro, such as the interferon-γ (IFN-γ) , tlie hepatocyte growth factor/scatter factor (HGF / SF) , the Oncostain M(OSM) , and the tumor necrosis factor-α (TNF-α)which is not produced by HIV-1-infected T cells. Treated and untreated BC-3 cells were collected at the 3rd and 7th day of persistent stimulation, respectively. Immuno-histochemical (IHC) staining, Northern blot, quantitative PCR (real- time PCR ) and electron microscopy (EM) were carried out to detect the expression of immunogenic protein ORF59, messenger RNA (mRNA) of minor capsid protein ORF26, and the presence of viral particles of HHV-8 from treated and untreated BC-3 cells. Results: It showed that IFN-γ, HGF/SF, OSM, and TNF-α were found to induce an increase in mRNA expression of ORF26 when added individually to BC-3 cells. Particularly, ORF26 expression stimulated with IFN-γ and TNF-α respectively, increased 6. 1 and 2. 5-fold(from real-time PCR results)at the 7th day when compared with untreated BC-3 cells. Meanwhile, about 20% of IFN-γ stimulated BC-3 cells expressed ORF59 at the 7th day as compared with 1. 5% of untreated BC-3 cells when IHC staining was employed. In addition, viral particles of HHV-8 were readily identified in BC-3 cells stimulated with IFN-γ at the 7th day with EM analysis. Conclusion;TNF-α and recombinant cytokines being similar to those produced by HIV- 1 infected T Cells could really induce HHV- 8 lytic cycle replication in BC-3 cells, another cell line of PEL.

Role of the virion host shutoff protein in neurovirulence of monkey B virus(Macacine herpesvirus 1)

Monkey B virus(Macacine herpesvirus 1; BV) is noted for its extreme neurovirulence in humans. Since the vhs protein encoded by the UL41 gene has been shown to be a neurovirulence factor in the related human herpes simplex viruses, the role of the UL41 gene in BV neurovirulence was investigated. BV mutants were constructed that lacked the entire UL41 ORF(Δ41) or had the RNase active site mutated(Δ41A). Neither mutant shut off host protein synthesis, degraded β-actin mRNA, or prevented an IFN-β response, indicating that the vhs protein and its RNase activity are both necessary for these activities. Replication of both mutants in primary mouse cells was impaired and they exhibited a prolonged disease course in mice. Whereas Δ41 infected mice were euthanized for symptoms related to central nervous system(CNS) infection, Δ41A infected mice were euthanized primarily for symptoms of autonomic nervous system dysfunction. While neuroinvasiveness was not affected, lesions in the CNS were more limited in size, anatomical distribution, and severity than for wild-type virus. These results indicate that the vhs protein affects the general replicative efficiency of BV in vivo rather than being a specific neurovirulence factor critical for invasion of or preferential replication in the CNS.

Regulation of Wnt/β-catenin signaling by herpesviruses

The Wnt/β-catenin signaling pathway is instrumental in successful differentiation and proliferation of mammalian cells. It is therefore not surprising that the herpesvirus family has developed mechanisms to interact with and manipulate this pathway. Successful coexistence with the host requires that herpesviruses establish a lifelong infection that includes periods of latency and reactivation or persistence. Many herpesviruses establish latency in progenitor cells and viral reactivation is linked to host-cell proliferation and differentiation status. Importantly, Wnt/β-catenin is tightly connected to stem/progenitor cell maintenance and differentiation. Numerous studies have linked Wnt/β-catenin signaling to a variety of cancers, emphasizing the importance of Wnt/β-catenin pathways in development, tissue homeostasis and disease. This review details how the alpha-, beta-, and gammaherpesviruses interact and manipulate the Wnt/β-catenin pathway to promote a virus-centric agenda.

The Biology of Kaposi’s Sarcoma-Associated Herpesvirus and the Infection of Human Immunodeficiency Virus

Kaposi sarcoma-associated herpesvirus (KSHV),also known as human herpesvirus 8 (HHV-8),is discovered in 1994 from Kaposi's sarcoma (KS) lesion of an acquired immunodeficiency syndrome (AIDS) patient. In addition to its association with KS,KSHV has also been implicated as the causative agent of two other AIDS-associated malignancies: primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV is a complex DNA virus that not only has the ability to promote cellular growth and survival for tumor development,but also can provoke deregulated angiogenesis,inflammation,and modulate the patient’s immune system in favor of tumor growth. As KSHV is a necessary but not sufficient etiological factor for KS,human immunodeficiency virus (HIV) is a very important cofactor. Here we review the basic information about the biology of KSHV,development of pathogenesis and interaction between KSHV and HIV.

Human herpesvirus 6A induces apoptosis of HSB-2 cellsvia a mitochondrion-related caspase pathway

Apoptosis plays an important role in the pathogenesis of viral infections.In this study,we investigated the cell death processes during productive HHV-6A infection and the underlying mechanisms.Annexin V-PI staining and electron microscopy indicated that HHV-6A is a strong inducer of apoptosis.HHV-6A infection decreased mitochondrial transmembrane potential and led to morphological changes of mitochondria.The cell death was associated with activation of caspase-3 and cleavage of DNA repair enzyme poly （ADP-ribose） polymerase,which is known to be an important substrate for activated caspase-3.Caspase-9 was activated significantly in HHV-6A-infected cells,whereas caspase-8 was not activated obviously.Moreover,HHV-6A infection upregulated Bax and downregulated Bcl-2.This is the first demonstration of mitochondrion-mediated,caspase-dependent apoptosis in HHV-6A-infected cells.

Development of a sandwich ELISA for the detection of bovine herpesvirus type 1

Objective:To develop a standard enzyme-linked immunosorbent assay（ELJSA） for the detection of bovine herpesvirus type 1（BHV-1）.Methods:The assay was based on hyperimmune rabbit and guinea pig antisera raised against purified BHV-1.Polyethylene glycol precipitation and sucrose density gradient methods were adopted for viral concentration and purification.Antisera were raised using Freund’s adjuvant followed by extraction of IgG of high purity.Results: Optimum antisera dilutions as determined by titrations were chosen as 14 000,whereas the conjugate was used at 1:2 000 dilution.Using 95 clinical specimens,the ELISA test showed a sensitivity and specificity of 91.90%and 93.10%,respectively when compared to PCR.The cutoff value was fixed at 0.15490) and a P/N ratio of】1.30 indicated a significant positive reaction. Conclusions:The results have demonstrated that this ELISA could efficiently detect BHV-1 and can be used as an important diagnostic tool.

Nuclear Egress of Herpesviruses

Herpesviruses assemble and fill their capsids in the infected cell nucleus, and must then move this enormous macromolecular assembly across the nuclear membrane and into the cytoplasm. Doing so is a complex, multi-step process that involves envelopment of the capsid at the inner nuclear membrane and de-envelopment by fusion with the outer nuclear membrane. This process is orchestrated by viral proteins, but requires the modification of cellular structures and mechanisms including the nuclear lamina. In this review I summarize recent research on the mechanism of nuclear envelopment and the viral and cellular systems involved in its execution.

Bovine Herpesvirus 1 Protein bICP0 Represses the Transcription of bISG15 in Fetal Bovine Lung Cells

The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung （FBL） cells. Bovine Herpesvirus 1（BHV-1）, which is a viral pathogen of cattle, can infect FBL cells and induce cytopathic effects. Real-time PCR assays showed that BHV-l＇s infection could repress the basal or inducible transcription of bISG15 in FBL cells. It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis. Our previous work showed that bIRF-3 was the key factor in the stimulation ofbISG15 in FBL cells, so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed. The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3. Taken together, our work suggested that BHV-1 had some molecular mechanism to resist the cellular bISG15＇s antiviral functions.

Prevalence of human herpesvirus 8 infection in patients undergoing hemodialysis using nested-PCR

Objective:To study the prevalence of HHV-8 infection in patients undergoing hemodialysis.Methods:In this study,blood samples of 89 patients undergoing hemodialysis were collected.DNA was extracted from peripheral blood mononuclear cells and HHV-8 DNA was evaluated by nested-PCR.Results:Of total 89 patients,51(57.3%)were males and 38(42.7%)were females.The patients'age ranged from 24 to 90 years and the mean age was(57.5±1.4)years.HHV-8 DNA was found in 9 of 89(10.1%)peripheral blood mononuclear cell samples,8/51(15.7%)in males and 1/38(2.6%)in females(P=0.07).All patients who were positive for HHV8-DNA were more than 50 years old.Conclusions:This study shows high prevalence of HHV-8.Since hemodialysis patients are candidates for kidney transplantation and due to the possibility of HHV8-reactivation and its serious complications in immunocompromised patients,routine screening for detection of the virus should be implemented for all hemodialysis patients.

Molecular Cloning and Sequencing of a Specific cDNA Mapping to the Bam HI-I2 and -LFragments within the Inverted Repeats of Unique Long Region(IRL) from the Genom e of the Marek′s Disease Herpesvirus (MDV) Oncogenic Strain Beijing-1

Isolation and identification of a specific cDNA mapping to the BamHI I2 and L fragments from the inverted repeats of unique long region(IRL) in the genome of Marek′s disease herpesvirus (MDV) oncogenic strain Beijing 1 had been previously performed by us. In this study, the specific cDNA was cloned into phagemid vectors PUC118 and 119 on the basis of prefabricated two recognized sites in synthesized primers. Recombinants were further identified with restriction pattern, molecular hybridization, and DNA sequencing analysis. It was demonstrated that this cDNA with 720 base pair (bp) contained sequences including a potentially incomplete open reading frame (ORF) encoded a 238 amino acids (aa) predicted polypeptide which was significantly homologous not only to part of N terminus of meq, but also to that of XbaI ClaI subfragment of BamHI L. In accordance with these data, following results could be deduced:①the 720 bp cDNA represented a spliced transcript;②meq transcription could be extended from the right hand end of BamHI I2 to the adjacent BamHI L fragment;③the L region was transcribed in varying degrees\ in MDV induced lymphoblastoid tumors.

Angiogenesis,Kaposi’s Sarcoma and Kaposi’s Sarcoma-associated Herpesvirus

Tumor angiogenesis is the uncontrolled growth of blood vessels in tumors,serving to supply nutrients and oxygen,and remove metabolic wastes. Kaposi’s sarcoma (KS),a multifocal angioproliferative disorder characterized by spindle cell proliferation,neo-angiogenesis,inflammation,and edema,is associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV). Recent studies indicate that KSHV infection directly promotes angiogenesis and inflammation through an autocrine and paracrine mechanism by inducing pro-angiogenic and pro-inflammatory cytokines. Many of these cytokines are also expressed in KS lesions,implicating a direct role of KSHV in the pathogenesis of this malignancy. Several KSHV genes are involved in KSHV-induced angiogenesis. These studies have provided insights into the pathogenesis of KS,and identified potential therapeutic targets for this malignancy.